Polypeptide having an action on the immunological system, process for its isolation and purification, its use, agents containing this compound, and its cleavage products, their use and agents containing these products

ABSTRACT

The invention relates to a polypeptide which can be isolated from thymus glands and has an action on the immunological system, and which has a molecular weight of 3,480, a UV absorption with a maximum at 205 to 210 nm and an isoelectric point of 3.95+0.15 and a specific aminoacid composition, the N-terminal, if appropriate, carrying an acyl group or an acylglycyl group, processes for its isolation and purification, its use, agents containing this polypeptide, and its cleavage products, their use and agents containing them.

The invention relates to a hitherto unknown polypeptide which can beisolated from thymus glands and has an action on the immunologicalsystem, and which has a molecular weight of 3,480, a UV absorption witha maximum at 205 to 210 nm, an isoelectric point of 3.95±0.15 and thefollowing aminoacid composition: 4 asparaginic acid units, 3 threonineunits, 3 serine units, 6 glutamic acid units, 1 glycine unit, 4 alanineunits, 3 valine units, 1 isoleucine unit, 1 leucine unit, 5 lysine unitsand 1 arginine unit, the N-terminal, if appropriate, carrying an acylgroup having 1 to 6 atoms or an acylglycyl group, the acyl group ofwhich has 1 to 6 carbon atoms, and to its peptide cleavage products andpharmacologically acceptable salts.

It is known that cell-free protein extracts from thymus glands of calves(for example thymosin fraction 5, which is a known standard product) canbe used to suppress immunological deficiency symptoms which, forexample, can cause reduced graft rejection, increased probability ofoncogenesis, increased susceptibility to infection, and acceleratedaging processes. The successful clinical use of this fraction 5, inparticular on lung cancer patients, has been reported (P. B. Chretien etal. Cancer Treat. Rep. 62 (1978) 1787-1790).

For obvious reasons, it is desirable in a therapy, instead of a mixtureof substances which possibly also contains inhibitors of the desiredeffect or even toxic compounds, to use characterized pure substanceshaving a defined and reproducible activity. Furthermore, the compositionof such a mixture of substances is difficult to standardize, since thecomposition depends, inter alia, on the age of the animals slaughtered.Examples of such pure substances which have been isolated from mixturesare thymosin α₁, β₁, β₄, β₈ and β₉, the first of the substancesmentioned having been the most thoroughly investigated with regard toits biological action (T.L.K. Low et al. J. Biol. Chem. 254 (1979)981-986, T.L.K. Low et al. Proc. Natl. Acad. Sci. USA 78 (1981)1162-1166).

Very recently, however, reports have appeared in particular on thymosinα₁ and β₈ (E. Hannappel et al. Proc. Natl. Acad. Sci. USA 79 (1982)1708-1711), and the present work on the isolation of the peptideaccording to the invention supports these findings that both peptidesresult only as a consequence of an isolation technique which is not mildand therefore difficult to reproduce (thymosin α₁ could not be detectedin secondary fractions in the isolation of the hormone according to theinvention).

It was therefore the object of the invention to isolate pure hormonesunder mild conditions in order thereby to retain the intact naturalactive principles. Since there is a large number of such activeprinciples in the thymus gland, and this plurality is certainly ofsignificance, for example with regard to varying specificity of action,the purification of all these substances is of the greatest importancefor providing an optimum spectrum for the therapy of the abovementioneddisorders.

The peptide isolated according to the invention has a high purity,preferably above 90%, as can be readily shown using methodsconventionally employed in peptide chemistry. These methods includepolyacrylamide gel electrophoresis, isoelectric focusing and aminoacidanalysis. The novel compound can be obtained in equally good yield andin the same high purity in different variants of the working upprocedure according to the invention, the products of all working upprocesses being identical to one another. This indicates that it is anintact natural substance and not a cleavage product dependent on theworking up procedure.

The peptide according to the invention is white, odorless and stable inneutral solution at elevated temperature, for example 80° C., and can bestored in the dry form at temperatures below +5° C. for at least 3 yearswithout loss in activity. From its chemical structure, it is apolypeptide in which, after complete hydrolytic cleavage, no otherconstituents can be detected apart from the aminoacids.

If the aminoacid composition is determined by the method of Moore andStein (Methods of Enzymology Volume VI, 819-831, published by Colovickand Kaplan, Academic Press, New York, London, 1963), the followingvalues are found: 4 asparaginic acid units, 3 threonine units, 3 serineunits, 6 glutamic acid units, 1 glycine unit, 4 alanine units, 3 valineunits, 1 isoleucine unit, 1 leucine unit, 5 lysine units and 1 arginineunit.

A characterizing feature of the polypeptide according to the inventionis the absence of methionine, cysteine, histidine, phenylalanine,tyrosine and tryptophan. The absence of, in particular, methionine andthe aromatic aminoacids can be used in the concentration processes forevaluating the purity of the fractions. It also provides the additionalexplanation for the fact that no exact correlation exists between thepeak areas in the chromatographic separation and the amount of substanceisolated from the peak when detection is effected at the conventionallyused wavelength of 254 nm. SDS-polyacrylamide gel electrophoresis (J. O.Thomas in: Techniques in Protein and Enzyme Biochemistry B 106, 102 etseq. (1978) Elsevier/North Holland, Biomedical Press) is used for themolecular weight determination, a value of about 3,000-4,000 beingobtained when the migration zone of the polypeptide is compared withthat of known reference substances, such as, for example, trypsininhibitor from soyabean (molecular weight 6,000) and insulin B-chain(molecular weight 2,500). This result correlates with the findings ofthe aminoacid analysis.

In basic disc electrophoresis in polyacrylamide gel (L. Ornstein, Ann.N.Y. Acad. Sci. 121 (1964) 321-349 and B. J. Davies, Ann. N.Y. Acad.Sci. 121 (1964) 404-427), the product according to the inventionessentially shows a single band with a Rf value of 0.5 to 0.6.Isoelectric focusing in 0.3 mm thick gels which have been prepared bythe technique of Radola (B. J. Radola, Elektrophoresis 1 (1980) 43-46)and contain ampholytes for the pH range 2-9 and 6M urea shows a similarpattern. The isoelectric point of the polypeptide according to theinvention, i.e. 3.95±0.15, is ascertained by comparison withcommercially obtainable test mixtures; the error range given resultsfrom the fact that the sample and comparison never have exactly the sameionic strength, and even the urea necessary for obtaining highresolution can influence the isoelectric point of polypeptides. Suchpolyacrylamide gels are stained with a modified Coomassie blue(Blakesley and Boezi, Analytical Biochemistry 82 (1977) 580-582).

The invention furthermore relates to a process for isolating thispurified polypeptide, which comprises isolating the polypeptide from thethymus with the aid of a combination of precipitation methods, membranefiltrations and chromatography.

The thymus glands are preferably extracted with an aqueous solution ofan inorganic salt having a buffering action in the pH range from 5.0 to9.0, preferably phosphate. The insoluble material is removed and thepolypeptide is separated off from the resulting extract and isolated.

The chromatography step of the above isolation process preferablycomprises cation and/or anion exchange chromatography, chromatography onnon-specific adsorbants, in particular hydroxyapatite, partionchromatography, in particular partition between two immiscible media,one of which contains a large amount of water, or a combination of twoor more of these methods.

It is advantageous to use for this chromatography an extract which hasbeen subjected to heat-denaturing, the peptide according to theinvention remaining in solution and retaining its complete activitywhilst other proteins can be separated off by sedimentation orfiltration. A material which is separated off as a precipitate ontreatment of the extract by addition of water-miscible organic solvents,preferably acetone, can also be used for the chromatography. Similarly,it is possible to use an extract which has been subjected to fractionalprecipitation by addition of water-soluble inorganic and/or organicsalts, preferably ammonium sulfate, the peptide precipitating in certainconcentration ranges, which depend on the salt used, and beingconcentrated by sedimentation techniques. An extract which, as a resultof fractionation by means of membrane filtration and/or gel filtration,still contains only peptides having a molecular weight below 10,000Daltons can furthermore be used for the chromatography.

Extracts which have been obtained by a combination of two or more of theprocesses described above could of course also be used for thechromatography.

The starting material for the preparation of the polypeptide accordingto the invention consists of fresh thymus glands or such gland materialwhich has been stored in a deep-frozen state promptly after slaughter.An aqueous extract is prepared from this material by comminution in thefrozen state and addition of a slat solution, with further cooling (0°to +4° C.). It is advantageous to subject this extract to an optimumtemperature of between 70° to 90° C., preferably 80° C., by rapidheating, because most of the proteases are thereby inactivated andseparated off as a precipitate. Separation is effected by centrifugationand filtration over gauze.

Comparison with other protease inactivation methods, such as theaddition of chemical inactivators, for example phenylmethylsulfonylfluoride (PMSF) has shown that neither the yield nor the activity dropssolely by heat treatment, and that this process is thus adequate per sefor this purpose.

In order to obtain smaller volumes and hence to facilitate furtherworking up, the protein fraction of interest should be concentrated outof the heat-treated extract by precipitation. This precipitation can becarried out by adding the extract to a water-miscible organic solvent.For example, several times the extract volume, preferably 5 times theamount, of acetone can be used, the precipitation being carried out atvery low temperatures of between -10° and -30° C., preferably at -20° C.It is also important to filter or centrifuge the mixture as rapidly aspossible, since other concomitant substances are also precipitated ifthe mixture if left to stand for a prolonged period. In this step also,enzymes possibly remaining in solution are inhibited by the highconcentration of organic solvent. The precipitate thus obtained can bestored in the frozen state without loss of activity.

Another possibility of concentration by precipitation, which can becarried out either instead of or after the abovementioned method, is theaddition of neutral salts, such as, for example, ammonium sulfate. Theprecipitation can achieve a certain selectivity by pH control. Byaddition of ammonium sulfate to 50% saturation, the peptide according tothe invention, which has an isoelectric point of about 3.95, can beprecipitated in the pH range between 3 and 5, preferably at pH 4, suchthat a large number of concomitant proteins remain in solution. Thisprecipitation is also carried out with cooling, preferably at between 0°and +4° C.

Two methods have proved suitable for separating off other proteinshaving a similar isoelectric point but a higher molecular weight: gelfiltration and ultrafiltration, the latter being particular suitable onan industrial scale. Gel filtration permits division into narrowermolecular weight ranges, but is of more limited capacity thanultrafiltration, so that it seems more suitable for later steps in thecourse of working up relatively large amounts of extract, for examplesubsequent purification of ultrafiltration or column chromatographyfractions. Spherical particles based on crosslinked dextran,polyacrylamide or modified silica gel, for example Bio-Gel®, Sephadex®,Ultrogel® or a polyol having a fractionation range of up to 10,000Daltons, are best used for the gel filtration. Because of theisoelectric point of the product according to the invention, the gelfiltration, as is in fact the case with all further purification steps,is best carried out in aqueous buffers at pH values between 4 and 9,since otherwise the solubility of the polypeptide is not ensured.

With two exceptions (CM-cellulose and hydroxyapatite, see below), the pHrange of 7.5-8.6 (ammonium bicarbonate or tris/HCl) has proved to beparticular advantageous.

The ultrafiltration is best carried out in two stages so that themembranes are not too heavily loaded (blockage of the pores). It hasproved advantageous, for example, to use a capillary membrane having anexclusion limit of 50,000 Daltons in combination with a flat membranehaving an exclusion limit of 10,000 Daltons. Rapid removal ofhigher-molecular material which would reduce the flow through the moreselectively operating flat membrane is achieved with the capillarymembrane.

On the basis of the method hitherto used, the material thus obtainedstill consists of a mixture of polypeptides. Because of its isoelectricpoint, the polypeptide according to the invention does not bond to acation exchanger at the conventionally used pH range of 4 to 5, so that,with the aid of these exchangers based on cellulose, dextran,polyacrylamide, polystyrene resin or silica gel, it can rapidly beseparated off from a large amount of the concomitant products which havean isoelectric point above pH 4 to 5 and are therefore absorbed. Thisseparation can be effected in a chromatography column or in a batchprocess.

In contrast, the peptide is bonded to an anion exchanger based ondextran, polyacrylamide, cellulose, polystyrene resin or silica gel atpH values of 5 to 9, preferably 8, and can be eluted substantiallyselectively by choosing suitable conditions. Examples of such conditionsare application of a pH or salt gradient. It has proved advantageous touse a 50 millimolar tris-HCl buffer, pH 8, and a sodium chloridegradient of 0 to 0.5M distributed over 5 to 10 times the column volume.Before this chromatographic separation, the polypeptide mixture shows alarge number of bands, which can scarcely be resolved, in basic gelelectrophoresis; staining with Coomassie blue gives the impression of acontinous background color with only very few defined bands. At thisstage, the polypeptide according to the invention is not visible. Aclear picture is obtained only on analysis of the ion exchangerfraction. Almost every peak of the chromatography can be correlated witha definite band of the electrophoresis, so that localization of thepeptide according to the invention is possible at about 35% of thegradient described in Example 7 (cf. FIG. 2).

Because of the complexity of the chromatogram, achievement of completehomogeneity in this first chromatography step is not ensured. A goodfurther purification effect can be achieved by rechromatography over thesame separation system. Gel filtration also separates off a furthersmall amount of higher-molecular material. However, the degree of purityis only slightly increased in this case, since the material has alreadypassed intensively through this separation principle (ultrafiltration).

It is advantageous to use a completely different method, i.e. absorptionon hydroxyapatite, before or after the rechromatography on ionexchangers, since this process is carried out with small losses andrapidly because the polypeptide according to the invention flows throughthe separating column unretarded in the conventionally used 10millimolar phosphate buffer at pH 6.8, and only concomitant substancesare bonded (cf. FIG. 3).

Chromatography on hydroxyapatite is generally included before the finalpurification by rechromatography on an anion exchanger, but this methodcan also be used with equal success at another point in the working upscheme (for example after the cation exchanger). The rechromatographystep on an anion exchanger is carried out in a column of low volume andhaving a substantially flatter gradient course (cf. FIG. 5).

It is self-evident that demineralization and lyophilization follow eachpurification step. Only the material obtained from the cation exchangertreatment can, since its salt content is low, be used directly for thenext step. The salts are removed either by ultrafiltration over asuitable flat membrane having an exclusion limit of 500 Daltons or10,000 Daltons, or by gel filtration over a suitable material based oncrosslinked dextran or polyacrylamide, for example Sephadex G 10 or G25, Bio Gel P-2 or Ultrogel ACa 202.

Buffers which can be removed during subsequent lyophilization, forexample ammonium bicarbonate, are of course best used.

The polypeptide according to the invention, the N-terminal of which ispresent as the free base, can be converted into the corresponding N-acylor N-acylglycyl derivative in a manner which is known per se, acylpreferably being Boc or C₁ -C₆ -alkanoyl, in particular acetyl.

Pharmacologically acceptable salts of the peptide according to theinvention are understood as meaning, in particular, alkali metal andalkaline earth metal salts, but also physiologically acceptable saltswith amines, such as alkylamines and cycloalkylamines.

The peptide according to the invention has been found to be active invarious conventional in vitro tests in respect of an action whichinfluences the immunological system. These include T celldifferentiation, the extent of which is measured by the E-rosette test(Aiuti, Scand. J. Immunol., 3, 521-532, 1974) and the phytohemaglutininstimulation test (PHA stimulation test).

In the spontaneous rosette test, the naturation of lymphocytes fromhuman umbilical cord blood is effected by rosetting with sheeperythrocytes in the cold. Since only mature T cells form rosettes in thecold with sheep erythrocytes, this is a specific T cell test, i.e. itenables observation of the shift of T cells (so-called null cells, arelatively large number of which occur in umbilical cord blood) to R⁺ -Tcells, i.e. mature T cells.

Because of the larger number of null cells, the normal value ofrosette-forming cells from umbilical cord blood is lower than in theperipheral blood of adults. A low rosette number can be correlated witha low immune on the basis of investigations of the blood of patientssuffering from autoimmune diseases or tumors. In the PHA stimulationtest (PHA=phytohemaglutinin) or the lymphocyte transformation test,conclusions about the number of mature lymphocytes, i.e. lymphocyteswhich can be stimulated, are obtained, not by investigation of thesurface structures, as in the spontaneous rosette test, but by thefunction test for ability to be stimulted with the plant lectin PHA,which bonds particularly to mature T cells.

The T lymphocytes are stimulated to blastogenesis by lectin in a waysimilar to that caused by antibacterial or viral antigens. This leads toproliferation, either directly or by the secretion of lymphokines. Theincorporation of radioactive thymidine within a certain time is then ameasure of the number of stimulated cells. Since only the mature orimmunologically potent lymphocytes are stimulated, the naturation, afterincubation with the substance, of lymphocytes worked up from umbilicalcord blood can be monitored by this test.

The peptide has also been recognised as particularly effective inrespect of an increase in the level of T lymphocytes in experiments withlymphocytes from immuno-deficient patients, for example with Di Georgesyndrome. The desired more powerful effect, which manifests itself in asubstantially slower effective concentration, of the pure substance incomparison with the mixture corresponding to "fraction 5" could also beclearly seen here (cf. Table 1).

                  TABLE 1                                                         ______________________________________                                                            Hormone according to the                                  "Fraction 5"        invention                                                 Dose μg/ml                                                                 125         250     500     5     10     100                                  ______________________________________                                        Activity*                                                                             311     244     111   133   322    88                                 ______________________________________                                         *Concentration of T lymphocytes in % in comparison with the standard          following incubation with fraction 5 or with the polypeptide according to     the invention.                                                           

Surprisingly, the full level of immunological activity is also retainedon fragmentation of the intact natural product by a chemical route or,preferably, with certain specific proteases, such as those havingtryptic or chymotryptic activity. Those peptidic cleavage products ofthe peptide according to the invention which contain 4 or moreaminoacids are preferred.

Fragment mixtures from digestion with TPCK trypsin or TLCK chymotrypsinhave been investigated intensively. The procedures with these twoenzymes are reproducible to the extent that, according toelectrophoresis, mixtures of the same composition are always formed.

The invention relates to the use of the peptide according to theinvention, its derivatives and/or its cleavage products produced by achemical or enzymatic route, for regulating or stimulating theimmunological system, as a therapeutic or prophylactic agent.

The invention furthermore relates to medicaments having animmunostimulating or immunoregulating action which can be used for thetreatment and prophylaxis of virus infections, immunodeficiencydiseases, oncogenesis (in particular carcinoses) and forms ofaccelerated aging processes and contain the polypeptide according to theinvention and/or its N-acyl derivatives and/or at least one of itsfragments in the free form or in the form of pharmacologicallyacceptable salts. In the therapy of malignant tumors, the peptidesaccording to the invention can also be used in combination withcytostatic agents (cf., for example, Mutschler, Arzneimittelwirkungen[Drug Actions], Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart,1981, pages 599-608) and/or radiation measures.

The polypeptides according to the invention can be used parenterally(for example subcutaneously or intravenously) or via mucous membranes(for example nasally or enterally).

The dosage for parenteral administration is 0.01-50 mg/m² of bodysurface/day, depending on the type of disease. In serious cases, it canalso be increased, since no toxic properties have yet been observed. Inparticularly serious cases, several administrations per week over aperiod of several weeks are recommended. As a result of the particularabsorption properties, doses for nasal administration must becorrespondingly higher.

For subcutaneous, intravenous or nasal administration, the compoundsaccording to the invention or physiologically acceptable salts thereofare converted into a solution, suspension or emulsion, if desired withthe conventional substances for this conversion, such as solubilizingagents, emulsifiers or other auxiliaries. Examples of possible solventsfor the novel active compounds and the corresponding physiologicallyacceptable salts are: water, physiological saline solutions or alcohols,for example ethanol, propanediol or glycerol, as well as sugarsolutions, such as glucose or mannitol solutions, or a mixture of thevarious solvents mentioned.

Parenteral administration can also be effected, for example, withexternal or implanted metering devices, such as, for example, automaticpumps, or in the form of a continuous drip.

In this case, it is advantageous for additives which stabilize againstdenaturation, such as are known, for example, from EP-A 18,609, to beadded.

The compounds according to the invention can also be administered in theform of a medicament with a depot action. In the case of injectionsolutions, such a depot action can be achieved, for example, bydissolving or suspending the medicament in an oily vehicle, addingmacromolecules which increase the viscosity and which delay thediffusion of the dissolved medicaments, absorbing the medicament ontosuitable carrier molecules, for example aluminum hydroxide, or usingcrystal suspensions.

For an oral use form, the active compounds are mixed with theconventional additives for this, such as excipients, stabilizers orinert diluents, and are converted into a suitable administration form,such as tablets, dragees, push-fit capsules, alcoholic or oilysuspensions or aqueous, alcoholic or oily solutions, by conventionalmethods. Examples of inert excipients which can be used are gum arabic,magnesium carbonate, potassium phosphate, lactose, glucose or starch, inparticular corn starch. Formulation can be effected as either drygranules or moist granules. Examples of possible oily excipients orsolvents are vegetable and animal oils, such as sunflower oil or codliver oil.

An aqueous sterile solution of the polypeptide according to theinvention, its derivatives and/or at least one of its fragments cancontain agents for rendering the solution isotonic, such as, forexample, about 0.9% strength sodium chloride or 1.7% strength glycerol,and, if appropriate, also preservatives, such as hydroxybenzoic acid orphenol.

EXAMPLE 1

500 g of frozen thymus glands are homogenized with 1.5 liters ofice-cold 0.09% strength sodium chloride solution and 5 ml of octanol ina mixer in the course of 3 minutes. After centrifugation at 0° C. and10,000 rpm for 30 minutes, the still cloudy supernatant liquor isclarified further by means of filtration over 2 layers of gauze, and isthen heated to 80° C. in the course of 15 minutes, while stirring. Theresulting precipitate is separated off by filtration over 4 layers ofgauze. The filtrate is rapidly cooled down to ±4° C. by stirring in anice-bath, and is run into 7.5 liters of acetone pre-cooled to -20° C.Another precipitate is formed, and is filtered off on a glass suctionfilter after 5 minutes and rinsed with 1 liter of cold acetone (-20°C.). After drying in vacuo, 6.1 g of a slightly yellowish powder with aprotein content (determined by the Lowry method) of 32% result.

EXAMPLE 2

25 kg of thymus in the frozen state (starting temperature -30° C.) areprecomminuted in a crushing mill and ground in a mincer (a total of 15minutes) and are then homogenized with 75 liters of 0.9% strength sodiumchloride solution, prepared from 74.3 kg of pyrogen-free water, 675 g ofsodium chloride and 0.75 g of phenylmethylsulfonyl chloride, at 0°-4° C.for 5 minutes in a 100 liter spray kettle. 3 rotor fillings of solid (15kg moist weight) are separated off via a tube centrifuge (Cepa®), cooledto 0°-4° C., at 15,000×g, in the course of 1 hour, and are discarded.83.6 kg of supernatant liquor are filtered over gauze, the filtrate iswarmed to an internal temperature of +80° C. in a 100 liter spray kettlein the course of 20-25 minutes, while stirring, and the resultingprecipitate (3 kg moist weight) is separated off in the course of 15minutes by means of a cooled tube centrifuge at 15,000×g. The slightlyopalescent supernatant liquor is filtered over 4 layers of gauze and isintroduced into 375 liters of acetone, precooled to -20° C., whilestirring. After 2 minutes, the resulting precipitate is centrifuged offvia a cooled tube centrifuge (15,000×g, 15 minutes). The centrifugeresidue (500 g moist weight) is washed with 3 liters of acetone of -20°C., centrifuged off in a beaker centrifuge at 3,000×g in the course of10 minutes and dried in a vacuum desiccator. 325 g of a slightlyyellowish powder with a protein content of 25% result.

EXAMPLE 3

250 g of acetone powder according to Exampe 1 or 2 are dissolved in 2.5liters of water and the solution is brought to pH 8.0 with 5N ammoniumhydroxide and stirred at 0°-+4° C. for one hour. The insoluble content(about 50 g after drying) is centrifuged off with a beaker centrifuge at5,000 rpm in the course of 30 minutes. After determination of the amountof protein by the conventionally used Lowry method, the supernatantliquor is adjusted to a protein content of about 25 mg/ml by adding 0.7liter of water, 1 liter of saturated ammonium sulphate solution is addedand the mixture is stirred at +4° C. for 1 hour. A precipitate forms andis separated off by centrifugation (5,000 rpm, 30 minutes). 628 g ofammonium sulfate are added to the clear supernatant liquor and the pH isbrought to 4.0 with glacial acetic acid. After the mixture has beenstirred at 0-+4° C. for 1 hour, the resulting suspension is againsubjected to centrifugation. The precipitate thereby obtained containsabout 35 g of protein, determined by the Lowry method, in addition to asubstantial amount of ammonium sulfate, and is therefore not dried, butused directly for the ultrafiltration.

EXAMPLE 4

The ammonium sulfate precipitate obtained from 250 g of acetone powderas described in Example 3 is dissolved in 1.5 liters of 0.1N ammoniumbicarbonate (pH 8.0) to give a clear solution, and is subjected toultrafiltration in an Amicon hollow fiber apparatus with 2 filtercartridges of the type H 10×50 (exclusion limit of 50,000 Daltons). Thesolution is thereby concentrated down to 300 ml, being replenished withtwo 500 ml portions of 0.1N ammonium bicarbonate (pH 8.0) towards theend of the ultrafiltration in order to achieve complete removal of allthe proteins below 50,000 Daltons. 19.8 g of residue containing all thehigher-molecular proteins is obtained after freeze-drying, the residuehaving a protein content, measured by the Lowry method, of 84%.

The filtrate is subjected to renewed ultrafiltration, this time in a 2.5liter Amicon cell with a 5PM10 flat membrane (exclusion limit of 10,000Daltons). The solution is thereby concentrated to 150 ml, beingreplenished with two 400 ml portions of 0.1N ammonium bicarbonate (pH8.0) towards the end. Freeze-drying of the residue gives 8.2 g with aprotein content of 89%.

A column 21.5 cm in diameter and 100 cm in length, filled with SephadexG10 (coarse) and equilibrated with 0.1M ammonium bicarbonate (pH 8.0) isused to free the filtrate from salts, in particular ammonium sulfate. Itis operated at a flow rate of 5 liters per hour. After about one thirdof the column volume, salt-free protein appears in the eluate, followedby a mixed fraction of protein and salt (protein content, measured bythe Lowry method, 48%), which is subjected to rechromatography over thesame column. 5.9 g of a material with a protein content of 93% areobtained in this manner, after freeze-drying.

EXAMPLE 5

17 g of a protein fraction obtained by ultrafiltration according toExample 4 are stirred with 500 ml of 0.1M sodium acetate buffer (pH5.5). An insoluble residue (1.15 g after drying) is separated off bycentrifugation (20 minutes, 5,000 rpm). 3 liters of CM-cellulose(Servacel CM-23), equilibrated with the same buffer, are added to thesoluble content. After the mixture has been stirred for half an hour,the suspension is passed through a glass suction filter and the cationexchanger is filtered off with suction, until almost dry, and rinsedslowly with 2 liters of the same buffer.

The filtrate (2.5 liters) is freeze-dried and dissolved in 150 ml of0.1M ammonium bicarbonate (pH 8.0), and the solution is demineralized,at a flow rate of 150 ml per hour, over a column 5 cm in diameter and 40cm in length, filled with Sephadex G10 (coarse) and equilibrated with0.1M ammonium bicarbonate (pH 8.0). In this case also, a fractioncontaining salt-free protein is initially obtained, followed by a mixedfraction of protein and salt, which is rechromatographed again over thesame column. 9.1 g of a material with a protein content of 94%, calledthe fraction in the following text, are thus obtained, afterfreeze-drying.

EXAMPLE 6

30 g of a protein fraction obtained by ultrafiltration according to aExample 4 are stirred into 1 liter of 0.01M sodium acetate (pH 5.5), andan insoluble content (1.3 g after drying) is separated off bycentrifugation (20 minutes, 5,000 rpm).

The clear solution is subjected to chromatography over a column 10 cm indiameter and 43 cm in length, filled with CM-cellulose (Servacel CM-32)and equilibrated with 10 liters of 0.01M sodium acetate (pH 5.5), thecolumn first being eluted with 10 liters of the same buffer at a flowrate of 500 ml per hour, after the sample has been applied. A peak (cf.FIG. 1) is thereby obtained, and is freed from salt components asdescribed in Example 5. 17.5 g of a fraction (in the followingdesignated as "CM1") with a protein content of 95% result fromfreeze-drying.

The column is washed free for renewed use by changing the buffer to0.01M sodium acetate and 1.0M sodium chloride (pH 5.5). A further peak(cf. FIG. 1) is thereby obtained and, after demineralization in theconventional manner and freeze-drying, gives 11.5 g of protein.

EXAMPLE 7

A column 10 cm in diameter and 62 cm in length is filled withDEAE-Sephacel (Pharmacia), which has been preswollen in a buffer of0.05M tris/HCl (trishydroxymethyl-aminomethane), and is equilibratedwith 15 liters of the same buffer at a flow rate of 325 ml per hour.

17.5 g of CM1 are dissolved in 1 liter of 0.05M tris/HCl (pH 8.5) andthe solution is pumped onto the column at 325 ml per hour. At the samerate, the column is rinsed with 1 liter of the same buffer and, finally,a linear gradient of 0 to 1.0M sodium chloride is applied over a volumeof 36 liters. A large number of small peaks, which overlap to give alarge main peak, become visible by detection at 245 nm (cf. FIG. 2).Each of these peaks is demineralized in the conventional manner andanalyzed by basic polyacrylamide gel electrophoresis (PAGE). From thisanalysis, the substance sought is found, with an Rf value of 0.5-0.6, inthe third peak of the main peak (labelled 2 in FIG. 2), containing 1,158mg of protein.

Comparison of the PAGE of the peak containing the substance with that ofthe fraction eluted beforehand (cf. insert in FIG. 1) shows the scarcelyavoidable overlapping of the individual peaks in this stage of thepurification, making subsequent purification of the individual peaksalso unavoidable.

EXAMPLE 8

A column 2.6 cm in diameter and 25 cm in length is filled withhydroxyapatite (Bio Rad) suspended in 0.01M sodium phosphate buffer (pH6.8) and is equilibrated with 500 ml of the same buffer at a flow rateof 80 ml per hour.

400 mg of the fraction, containing the substance sought, from the anionexchange chromatography are dissolved in 50 ml of 0.01M sodium phosphatebuffer (pH 6.8) and a linear gradient of 0.01-0.40M sodium phosphate (pH6.8) is applied over 1.3 liters, with 80 ml of the same buffer (cf. FIG.3). The substance sought is in the breakthrough peak (peak 1 in FIG. 3)and is isolated from this, in a yield of 290 mg, together with someother polypeptides, by demineralization and freeze-drying carried out inthe conventional manner.

The resulting material shows a similar band spectrum to the startingmixture in the PAGE, but with a considerably higher intensity of theband corresponding to the polypeptide according to the invention.

EXAMPLE 9

A column 1.6 cm in diameter and 120 cm in length is filled with UltrogelACA 54 (LKB), preswollen in 0.1M ammonium bicarbonate buffer (pH 8.0),and is equilibrated with 750 ml of the same buffer at a flow rate of 30ml per hour.

290 mg of the fraction, containing the substance sought, from the anionexchange chromatography are dissolved in 30 ml ammonium bicarbonatebuffer (pH 8.0) and the solution is pumped onto the column at 30 ml perhour, the column then being further rinsed with the same buffer at thesame flow rate (cf. FIG. 4).

A small breakthrough peak containing small amounts of high-molecularproteins, and, after twice the volume, a broad main peak which gives thesame band spectrum in the PAGE throughout the entire volume, appear.

Freeze-drying of this main peak gives 200 mg of a material of onlyslightly better quality than the starting material.

EXAMPLE 10

A column 2.5 cm in diameter and 35 cm in length is filled withDEAE-Sephacel (Parmacia), preswollen in 0.005M tris/HCl (pH 0.8), isequilibrated with 500 ml of the same buffer at a flow rate of 60 ml perhour.

220 mg of the hydroxylapatite fraction containing the same substance isdissolved in 50 ml 0.05M tris/HCl (pH 8.0) and is pumped onto thiscolumn at a flow rate of 60 ml per hour. The column is then eluted, atthe same rate, with a linear gradient of 0 to 0.3M sodium chloride overa volume of 2 liters (cf. FIG. 5).

A main peak composed of several peaks appears approximately in themiddle of the gradient. If this is separated into 5 fractions (cf. FIG.5), the substance according to the invention is predominantly infraction 4. It can be isolated from this fraction in a yield of 70 mgand with a purity of over 90%, according to the PAGE, bydemineralization and freeze-drying carried out in the conventionalmanner.

EXAMPLE 11 Typical cleavage

An enzyme-substrate ratio of 1/100 at a concentration of 1 mg/ml ofsubstrate in 0.1M N-methylmorpholine acetate buffer, pH 8.1, is used inthe enzymatic cleavage. Incubation at 37° C. was carried out withTPCK-treated trypsin for 2 hours or with TLCK-treated α-chymotrypsin for7 hours. After acidification to pH 2.5 with 6N glacial acetic acid, thesolution was heated rapidly to 80°. After 2 minutes, the solution wascooled again rapidly and centrifuged. The supernatant liquor was thenlyophilized, whereupon a salt-free, white powder was obtained.

EXAMPLE 12 E-rossette test

In the test, which is carried out by the method of Aiuti (Scand. J.Immunol., 3, 521-532, 1974), the rosette counts oflymphocyte/erythrocyte systems are determined after incubation (37° C.)for 2 hours in the medium or in the medium+substance. In the assessment,of 200 cells counted, only those which consist of at least onelymphocyte and three erythrocytes are evaluated as rosettes.

The ratio of the number of rosettes after preincubation with substanceand without substance, the stimulation index S_(I), is then a measure ofthe differentiation of the null cells to mature T cells induced by thesubstance.

Results:

    ______________________________________                                                                 Polypeptide according                                Dose μg/ml                                                                           Thymosin fraction V                                                                          to the invention                                     ______________________________________                                        20        S.sub.I = 1.9  S.sub.I = 3.1                                        10        2.6            /                                                    5         2.0            2.5                                                  2.5       1.9            /                                                    1.25      1.3            2.7                                                  ______________________________________                                    

Whilst fraction V, as a mixture of various peptides of differentbiological activity, causes an increase in the rosette count from 17% toonly 22% at a concentration of 1.25 μg/ml, incubation of the lymphocyteswith 1.25 μg/ml of the peptide according to the invention leads to anincrease in the rosette count from 18.5% to 50.5%.

EXAMPLE 12a Phytohemaglutinin stimulation test

The substance(s) are introduced into the cell suspension in variousconcentrations (10⁵ cells in 100 μl of RPMI 1640/Click+Hepes 20 mM,penicillin; streptomycin glutamine and 10% AB-serum inactivated by heat)about 1 μCi/well of tritium-labelled thymidine is added either after 48hours or after 72 hours, the mixture is incubated for 8 hours, the cellsare filtered off with suction on glass fibre filters and washed and thefilters are later introduced into scintillation liquid and counted in aβ-counter.

Since the substance(s) is present throughout the entire duration of theculture, possible degradation or change by serum or lymphocyte proteasescannot be excluded.

    __________________________________________________________________________    Results:                                                                       Substance                                                                             PHA (μg/ml)Concentration                                                           (hours)Incubation time                                                                (μg)Dose                                                                      cpm                                                                               x.sub.1                                                                           Control                                                                            -x.sub.2                                                                          ##STR1##                         __________________________________________________________________________    Polypeptide                                                                           10      48      1  24,772  16,216   1.45                              according to               20,334                                                                            20,965   14,472                                the invention              17,791  12,730                                     Mixture of                                                                            10      48      1  19,257                                             fragments after            22,817                                                                            21,013       1.45                              tryptic cleavage           20,967                                                             48      2.5                                                                               7,994   7,468                                                                 9,166                                                                             8,621                                                                             6,815                                                                              7,731                                                                            1.12                                                          8,705   8,910                                                     72      2.5                                                                              14,606  12,177                                                                15,598                                                                            15,522                                                                            13,710                                                                             12,943                                                                            1.24                              Mixture of                                                                            20      72      1  37,624  25,247                                     fragments after            42,771                                                                            40,456                                                                            37,921                                                                             32,409                                                                            1.25                              chymotryptic               41,972  34,058                                     cleavage                                                                      __________________________________________________________________________

EXPLANATION OF THE FIGURES FIG. 1 The material obtained byultrafiltration is fractionated on CM-Cellulose

10×43 cm Servacel CM 32 in 0.1M sodium acetate, pH 5.5

Flow rate: 500 ml per hour

Quantity introduced onto: 30 g

After 10 l: exchange of the buffer (now 0.1M sodium acetate, 1M sodiumchloride, pH 5.5)

The insertion shows a picture of a basic electrophoresis onpolyacrylamide gel.

yields:

fraction I: 17.5 g

fraction II: 11.5 g

FIG. 2 Fraction I obtained above is fractionated on DEAE-Cellulose

10×62 cm DEAE-Sephacel in 0.05M

Tris/HCl, pH 8.5.

Flow rate: 350 ml per hour

Gradient of 0 to 1M sodium chloride over a volume of 36 l

Quantity introduced onto: 17.5 g

The insertion shows a picture of a basic eletrophoresis onpolyacrylamide gel.

yields:

fraction I: 722 mg

fraction II: 1,158 mg

FIG. 3 A fraction obtained from DEAE-Cellulose is purified onhydroxylapatite

2.6×25 cm hydroxylapatite in 0.01M sodium phosphate, pH 6.8

Flow rate: 80 ml per hour

Gradient of 0.01 to 0.40M phosphate (pH 6.8) over a volume of 1.3 l

Quantity introduced onto: 400 mg

The insertion shows a picture of a basic electrophoresis onpolyacrylamide gel.

yields:

fraction I: 290 mg

fraction II: 40 mg

fraction III: 9 mg

FIG. 4 Gel permetation chromatography of a fraction obtained fromDEAE-Cellulose

1.6×120 cm Ultragel ACA 54 in 0.1M ammonium hydrogencarbonate, pH 8.0

Flow rate: 30 ml per hour

Quantity introduced onto: 290 mg

The insertion shows a picture of a basic electrophoresis onpolyacrylamide gel.

yields:

fraction I: 2 mg

fraction II: 72 mg

fraction III: 128 mg

FIG. 5 Re-chromatography of fraction I, obtained from hydroxylapatite,on DEAE-Cellulose

2.5×35 cm DEAE-Sephacel in 0.05M Tris/HCl, pH 8.0

Flow rate: 60 ml per hour

Gradient of 0 to 0.3M sodium chloride over a volume of 2 l.

Quantity introduced onto: 220 mg

The insertion shows a picture of a basic electrophoresis onpolyacylamide gel.

yields:

fraction I: 28 mg

fraction II: 53 mg

fraction III: 42 mg

fraction IV: 70 mg

fraction V: 12 mg

We claim:
 1. A polypeptide having an action on the immunological system,which has a molecular weight of 3,480, a UV absorption with a maximum at205 to 210 nm, an isoelectric point at 3.95±0.15 and the following aminoacid composition: 4 asparaginic acid units, 3 threonine units, 3 serineunits, 6 glutamic acid units, 1 glycine unit, 4 alanine units, 3 valineunits, 1 isoleucine unit, 1 leucine unit, 5 lysine units and 1 arginineunit, the N-terminal, if appropriate, carrying an acyl group having 1 to6 carbon atoms or an acylglycyl group, the acyl group of which has 1 to6 carbon atoms, and/or pharmacologically acceptable salts.
 2. Apolypeptide as claimed in claim 1, which has a purity of more than 90%.